Method and a device for treating microparticles

ABSTRACT

Method for handling microparticles in such a manner, that at least two treatment steps are performed for microparticles in the same vessel without moving the particles to another vessel. There are organs in the device for changing the solution without having to move the microparticles to another vessel.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/300,347, filed Nov. 18, 2011, issued as U.S. Pat. No. 8,430,247;which application is a divisional application of U.S. application Ser.No. 10/576,297, filed Mar. 8, 2007, issued as U.S. Pat. No. 8,084,271;which application is a U.S. national stage application filed under 35U.S.C. §371 of International Patent Application No. PCT/IB2004/003433,accorded an international filing date of Oct. 20, 2004; whichapplication claims priority to Finnish Patent Application Nos.FI-20040159, filed Feb. 2, 2004, and FI-20031535, filed Oct. 20, 2003;which applications are incorporated herein by reference theirentireties.

FIELD OF THE INVENTION

The invention relates to a method for treating microparticles. Theinvention further relates to a device for treating microparticles.

BACKGROUND OF THE INVENTION

Magnetic transfer method refers to all action related to the movement ofparticles by means of magnetism, such as assorting, collecting,transferring, mixing and dosing within a solution or from one solutionto another.

Particles, microparticles or magnetic particles refer to all such smallparticles that have their diameter in the range of micrometers, and thatcan be moved by means of magnetism. There are various known particlesthat are transferable with a magnet and applications, where they areused, also greatly vary. For example, particles used in microbiologyusually have a size of 0.01-100 μm, most commonly 0.05-10 μm. Such knownparticles are, for example, particles containing ferromagnetic,paramagnetic or supramagnetic material. Particles can also be magneticthemselves, whereby they can be moved by means of any ferromagneticobject.

In a device intended to treat microparticles, there is a unit exploitingmagnetism, that is hereinafter referred to as a magnet. It can be apermanent magnet or an electrical magnet, that attracts ferromagneticparticles, or a ferromagnetic object, that is not magnetic itself, butstill attracts magnetic particles.

A magnet is usually preferably a rounded bar magnet. It can also be abar of another shape. However, a magnet does not need to be a bar atall. It can also be short and broad or an object of any shape. A magnetcan also consist of one or more objects, such as magnets orferromagnetic objects.

There has to be a shield covering the magnet, protecting the magnet fromvarious harmful conditions and enabling the treatment of microparticles,such as binding and release. The structure of the shield may greatlyvary, for it can be, for example, a thin membrane made of flexible orstretching material or even a cup made of rigid plastic.

Microparticles are usually used as the solid phase to bind variousbiomolecules, cell organelles, bacteria or cells. For example, enzymescan be immobilised on the surface of microparticles, whereby thetreatment and further use of the enzymes is efficient. Most of the socalled magnetic nanoparticles (<50 nm) are not suitable to be treatedwith regular permanent magnets or electrical magnets, but require theuse of an particularly strong magnetic gradient, as described in EP0842704 (Miltenyi Biotec). Magnetic particles, such as microparticles,that have a diameter of about 0.1 pm or more, can usually be treatedwith regular permanent or electrical magnets. The viscosity of thesolution can also considerably hamper the picking of the particles. Theparticles to be picked can be originally suspended in the solution,where a substance is desired to be bound, or, say, cells on the surfaceof the microparticles.

PRIOR ART

Microparticles treated by means of a magnet have been used since the1970's. This technique became very popular in immunoassays, amongothers. The use of microparticles in immunoassays to separate the boundantigen-antibody complex from the free fraction provided a considerableadvantage particularly in the reaction rate. The main developmentconcerning the use exploitation of microparticles has over the pastyears occurred in the field of molecular biology, microbiology and cellbiology.

In a classical method the magnetic particles present in the reactionsolution, such as microparticles, are captured by means of an externalmagnet to a given spot on the inner surface of the tube. Thereafter aneffort is made to carefully remove the solution around themicroparticles. In a classical method liquids are actively treated andmagnetic particles stay in the same vessel during the whole procedure.

In another approach a magnet is used to actively transfermicroparticles. The magnet is put in a solution containingmicroparticles, whereby the magnet attracts microparticles in thesolution and they form a solid precipitate. Thereafter the magnet andthe microparticles can be drawn out of the liquid. The magnet togetherwith its particles can thereafter be soaked in a liquid in another testtube, wherein the microparticles can be released from the magnet. Inthis method the treating, pipetting and aspiration phases are minimisedto the extreme.

U.S. Pat. No. 2,517,325 (Lamb) describes a solution for picking metalobjects by means of a magnet. The publication describes a long barmagnet, that is moved inside an iron tube. The poles of the bar magnetare at the opposite ends of the longitudinal axis of the physicalmagnet. By moving the magnet out of the iron tube, the magnetic fieldbecomes stronger. The publication describes a solution, wherein metalobjects can be collected to the tip of the magnet unit. The publicationalso describes a solid plastic shield for protecting the magnet.

U.S. Pat. No. 2,970,002 (Laviano) describes a solution for collectingmetal objects in liquids by means of a magnet. The publication describesa long permanent magnet, that collects particles to the tip of themagnet unit. The magnet is attached to the metal bar and protected witha separate plastic shield. The publication describes the simultaneoususe of moving of the permanent magnet and the plastic shield used toprotect the magnet. The publication describes the collecting of metalobjects to the tip of the magnet unit and the release of the metalobjects from the top of the shield by means of a particular design ofthe plastic shield.

U.S. Pat. No. 3,985,649 (Eddelman), U.S. Pat. No. 4,272,510 (Smith etal.), U.S. Pat. No. 4,649,116 (Daty et al.), U.S. Pat. No. 4,751,053(Dodin et al.) and U.S. Pat. No. 5,567,326 (Ekenberg et al.) describesolutions, wherein magnetisable material is collected directly from thesolution with a magnet in each of them. It is also common for thesepublications that the magnets are not protected with separate plasticshields. These solutions also require washing of the tip of the magnetbefore treating the next sample to eliminate the risk of contaminationand the carry-over effect of impurities.

U.S. Pat. No. 5,288,119 (Crawford, Jr. et al.) describes a solution,wherein metal objects can be collected by means of a magnet. The magnetof the device according to the publication is not protected with aparticular shield and it is not suitable for picking metal objects inliquids. The publication describes a solution for picking larger metalobjects. The publication shows a long bar magnet, that is moved inside anon-magnetic tube. A special characteristic of this tube is that it actsas a blocking of the magnetic field, although it is not magnetic. Asalternative materials for this purpose the publication shows, forexample, bismuth or lead or a mixture thereof. The magnet of the deviceaccording to the solution is not protected with a particular shield andit is not suitable for picking metal objects in liquids.

WO Application 87/05536 (Schröder) describes the use of a permanentmagnet, movable inside the plastic shield, for picking ferromagneticmaterial in a solution containing them. Ferromagnetic material gathersto the tip of the magnet unit, while the magnet is in its lowerposition. The publication describes the transfer of the ferromagneticmaterial collected in this manner into a solution in another vessel andthe release of the material there off the tip. The release of theferromagnetic material is described by means of the design of theplastic shield that prevents the material from moving while moving themagnet upwards.

U.S. Pat. No. 5,837,144 (Bienhaus et al.) describes a method forcollecting microparticles by means of a magnet equipped with aparticular plastic shield. This publication describes the binding ofmicroparticles in a solution, which is conducted out of the vessel bydifferent arrangements. By moving the magnet the microparticles can bemade to become free from the surface of the protective shield.

U.S. Pat. No. 5,942,124 (Tuunanen), U.S. Pat. No. 6,020,211 (Tuunanen),U.S. Pat. No. 6,040,192 (Tuunanen), U.S. Pat. No. 6,065,605 (Korpela etal.), U.S. Pat. No. 6,207,463 (Tuunanen) and US Patent Application20010022948 (Tuunanen) also describe devices equipped with a plasticshield for collecting microparticles in a solution and transferring themto another solution. These publications primarily describe solutions,where the intention is to treat microparticles in very little volumes.U.S. Pat. No. 5,942,124 (Tuunanen) describes a device, by means of whichmicroparticles can be enriched right to the tip of the magnet unit. U.S.Pat. No. 6,020,211 (Tuunanen) describes the use of the device presentedin the previous publication for transferring microparticles collectedtogether by means of a big, so called classical magnet to smallervessels. U.S. Pat. No. 6,040,192 (Tuunanen) describes an automatedmethod for using microparticles in specific assays and for handlingsmall volumes. U.S. Pat. No. 6,065,605 (Korpela et al.) continues tofurther apply the solution described in U.S. Pat. No. 5,942,124(Tuunanen) for handling fairly large volumes. Now a method, wherebymicroparticles have first been collected by means of a particular magnetunit containing a big magnet, is described. Thereafter the magnet unitdescribed in U.S. Pat. No. 5,942,124 (Tuunanen) is used for transferringthe pellet of microparticles further into smaller vessels. U.S. Pat. No.6,207,463 (Tuunanen) also applies the previously described magnet unit,by means of which magnetic particles can be collected right to the tipof the device. US Patent Application 20010022948 (Tuunanen) describesalso the treating of a very small amount of microparticles in particularvessels design for this purpose.

U.S. Pat. No. 5,942,124 (Tuunanen), U.S. Pat. No. 6,020,211 (Tuunanen),U.S. Pat. No. 6,065,605 (Korpela et al.), U.S. Pat. No. 6,207,463(Tuunanen) and EP 0 787 296 (Tuunanen) describe a method, where a largeamount of microparticles are intended to be collected from a fairlylarge vessel by means of a very small magnet to the small tip of a verysharp and narrow bar, the method being impractical.

U.S. Pat. No. 6,403,038 (Heermann) describes a device, that has aplastic shield and a permanent magnet attached to a particular bar.Microparticles are collected to the tip of the plastic shield and themethod is particularly intended for treating small volumes. The bar hasa particular, projecting part, by means of which the magnet and the barkeep still in the protective tube.

EP Patent 1058851 (Korpela) and Patent Application WO 01/60967 (Korpela)describe devices, that have a stretchy, elastomeric protective membrane.In these solutions the microparticles are collected on the surface ofthe stretchy protective membrane, where they can be further transferredto another vessel. The protective shield of the magnet is made ofstretchy material, whereby the membrane is as thin as possible whenstretched. In this way a distance as small as possible from the magnetto the liquid is brought about.

Currently known approaches require either transferring themicroparticles from one vessel to another containing a new solution orbinding the microparticles to the inner surface of the vessel whileremoving the previous solution and replacing it with a new one. Theprevious manner consumes a lot of vessels, because each new wash orincubation requires the use of a new vessel. In this method the use ofdisposable vessels is extensive and also the solutions concerningdevices require developing of a large device. The latter manner i.e. theso called conventional manner to treat microparticles has the problem ofcontrolling the magnetic field to be used and homogenisation of themicroparticles. The normal manner, for example, using 96-well plates, isto use a magnet plate, that has either magnet pivots or magnet bars ontop of the plate. The magnets on the magnet plate are placed in such amanner that they go into the spaces and gaps between the wells of the96-well plate to be used. While the 96-well plate is placed on top ofsuch a magnet plate, the magnets bring about a magnetic field in thewells of the 96-well plate and the microparticles in these wells attachto the inner surface of the well to form a layer of microparticles. Nowthe solution may be removed from the well, for example, by means of apipette and the next solution may be brought in. The microparticles needto be homogenised in the solution off the inner surface of the well andfor this purpose the 96-well plate needs to be removed from its positionon top of the magnet plate in order to disconnect the magnetic field.Homogenisation of the microparticles in the solution, mixing thesolution and the microparticles in the well as well as evaporation ofthe liquid are problems, not easy to solve, in the conventional method.These problems are particularly pronounced in small vessels, such as,for example, when using 96-, 384- and 1536-well plates.

According to one preferred embodiment the microparticles are bound onthe surface of a specific magnetic device and the solutions may bechanged through a hole on the bottom of the vessel. In this particularmethod, though, neither transferring of microparticles from one vesselto another, removing the microparticles temporarily away from the vesselwhile changing the solutions, mixing the solution and/or themicroparticles nor closing the vessel, is being solved.

None of the previously described publications describe a method, bymeans of which microparticles could be efficiently collected, mixed andincubated particularly in small volumes. Neither do the publicationsdescribe an efficient manner to transfer microparticles within onevessel, to transfer microparticles from the vessel nor to changesolutions when the need arises depending on the application.

Method

The purpose of this invention is to bring about such a method fortreating microparticles that does not have the previously presenteddisadvantages. It is characteristic for the method according to theinvention that at least two treatment steps are performed in the samevessel without moving the particles into another vessel.

The invention relates particularly to collecting microparticles,removing and adding solutions in one vessel, mixing microparticles inthe vessel, closing the opening of the vessel and transferringmicroparticles from one solution to another. By means of the methodaccording to the invention, operation of a large sample volume by meansof a vessel including a filter and coating of the magnet unit or itsprotective membrane with microparticles, may be performed. A coating,brought about in such a manner, may be used in, for example,purifications and immunoassays.

The method may be used particularly in automated devices, where varioustransfers, washes and incubations of microparticles may be performed. Itis possible to join the automated device to units, whose purpose is, forexample, to detect PCR reactions and/or various labels used inimmunoassays.

Device

It is characteristic for a device for treating microparticles that thereare organs in the device for changing the solution in such a manner,that at least two treatment steps for the microparticles can be done inthe same vessel without transferring the particles to another vessel.

The invention further relates to a device for mixing microparticles,closing of the opening of the vessel to be used, a specific mixingdevice, operating a large sample volume by means of a vessel including afilter and a magnet unit as well as its protective membrane.

The crucial property of the wash station for microparticles according tothe invention is that the microparticles do not need to be transferredto another vessel, but various incubations and washes may be performedin the same vessel. The solution may be changed in the vessel by, forexample, aspirating the solution away from the vessel through a filteron the bottom of the vessel or a specific channel. Aspirating thesolution may be performed, for example, by means of an aspiration orvacuum device, that is joined to the vessel to be treated.Alternatively, aspiration of the solution from the vessel may beperformed by means of a pipette or a washing device. Microparticles arebound on top of the protective membrane of the magnet unit or on theinner surface of the vessel to be used by means of an external magnetand a ferromagnetic sleeve, while changing the solutions. The newsolution may be brought into the vessel by pipetting or by means ofvarious dispensers/washers. One of the advantages of the wash stationfor microparticles according to the invention is that microparticles donot always need to be transferred to new vessels, while new solutionsare being introduced. In this way a lot of disposable plastic ware, suchas, for example, plates and tubes, can be saved and the size of theautomated devices may be elaborated to become remarkably small. It isparticularly easy to disconnect and connect the magnetic field, whenusing the wash station according to the invention.

One wash station according to the invention may be realised by means ofan external magnet and a ferromagnetic sleeve/plate. In this case theferromagnetic sleeve is used in an appropriate manner to remove orcreate the magnetic field in the vessel. Removing the solution from thevessel may be brought about by either aspirating the solution through afilter/membrane/channel on the bottom of the vessel or by means of apipette/washing device from the top of the vessel. Microparticles arecollected before removing the solutions either on the inner surface ofthe vessel to form a layer or on top of the protective membrane of themagnet unit. The next solution may be brought in by means of a pipetteor a dispenser/washer. In one embodiment of the invention, a specificmixing tool is brought into the vessel, which tool consists of a barinside of a protective membrane consisting of elastomer material, whichbar may be used to stretch or loosen the protective membrane. In thisway an efficient mixing is brought about in the solution inside thevessel. Mixing can also be arranged by means of the protective membraneof the magnet unit, whereupon the movement of the magnet and theferromagnetic sleeve inside the protective membrane in relation to eachother is exploited.

An external magnet and ferromagnetic sleeve/aperture plate may acttogether with the vessel in such a manner, that there is an appropriatespring under/on one side of the magnet. While resting, me magnet issituated outside the ferromagnetic sleeve/aperture plate by means of thespring and a magnetic field is directed towards the vessel. Whilepushing the magnet inside the ferromagnetic field, the spring yields andthere is no magnetic field directing to the vessel. A specific lockingmechanism may be arranged in the wash station in order to keep thespring yielded. It is possible to replace the spring with a specificmotorised solution, whereupon the magnet is moved under controllableconditions in relation to the ferromagnetic sleeve.

The opening of the vessel may be tightly closed by means of a magnetunit used together with a wash station according to the invention, forexample, during the incubations. This property is of great importancewhen using small volumes of solution, whereupon evaporation of thesolution is often a considerable problem. Also incubations at hightemperatures and long incubation times require closing of the vesselsduring the incubations. By means of tools according to the invention,solutions and/or microparticles can be mixed, while the vessel is closedwithout the help of an external mixer/shaker. In the mixing manneraccording to the invention, by stretching and loosening the protectivemembrane consisting of elastomer material, an efficient mixing may bebrought about in even small vessels. The protective membrane may haveappropriate forms to enhance the mixing efficacy.

Microparticles may be brought to form a layer of microparticles on thesurface of a filter or a membrane by means of the method according tothe invention. By means of such a method application of microparticles,for example, a large sample volume may be aspirated through the layer ofmicroparticles and the filter under it. The microparticles in the layerof microparticles may be coated, for example, with specific antibodies,to which the desired components in the sample are bound. Thereafter themicroparticles may be collected from the layer onto the surface of theprotective membrane by means of the magnet unit and transferred from thevessel including the filter. The components collected from the samplemay be released to an appropriate solution off the surface of themicroparticles or the microparticles may be processed further dependingon the application in question.

In one method according to the invention the microparticles are bound tothe protective membrane of the magnet unit to form a layer. The magnetunit and the solution containing the microparticles are mixedappropriately in order to make the layer on top of the protectivemembrane as homogenous and thin as possible. By using a transverselymagnetised magnet a very large area of the surface of the protectivemembrane may be taken into use to collect microparticles to form a thinlayer on top of the protective membrane. On the other hand, a magnetthat is magnetised along the longitudinal axis of the magnet unit, whichmagnet collects microparticles to the very tip of the protectivemembrane, is a preferred alternative, when a very large solid-phase areais not needed for the assay/purification, but the desired volumes ofsolution are very small. A large amount of microparticles is not themost important circumstance in this solution, but the fact, how largethe area and how homogenous the layer is, where the microparticles arearranged. A microparticle coating, brought about in such a manner, isparticularly preferred to be used, for example, when performing animmunoassay. It is desired in immunoassays, as well as in many otheranalytical applications, that the collecting solid-phase is as large aspossible, whereupon the sensitivity of the measurement may be increasedand the reaction kinetics may be enhanced. In this case themicroparticles are not to be released from the surface of the protectivemembrane, but all the incubations and washes are performed together withthe layer of microparticles. It is also secured by means of thissolution, that microparticles are not lost during the process comparedto the case, where the microparticles are released in each wash step tothe solution and gathered back from it after the wash step. Also in thiscase the wash station, mixing and closing of the opening of the vesselaccording to the invention may be utilised. In one embodiment allnecessary solutions and microparticles may be readily dispensedbeforehand in separate vessels and the vessels may also be closed. Suchan approach is preferred, when a very simple and ready-to-use method isdesired to be developed. In one embodiment according to the inventionthere is no separate shield or protective membrane around the magnet orthe magnets in the magnet unit; but the magnet may be coated inappropriate manner, for example, with a phosphate, epoxy or nickelcoating.

The magnet in the magnet unit according to a preferred embodiment of theinvention has the essential technical characteristic, that the strengthand the adjusting of the magnetic field can be regulated in relation tothe protective membrane surrounding the magnet. This can be broughtabout by moving the magnet inside the ferromagnetic tube in such amanner, that it can be completely inside the tube, whereupon theefficiency of the magnet is insignificant or nonexistent, or it can bepartially or completely outside the tube, whereupon the efficiency andthe collecting area of the magnet are in relation to the protruding partof the magnet. Combining these characteristics for transferring magneticparticles to vessels of appropriate sizes a very efficient collectingand enrichment event is brought about.

A ferromagnetic tube can consist of iron or other suitable material,which has appropriate characteristics to stop the magnetic stream fromgetting through the tube. The efficiency of the magnet can be regulatedby changing the place of the magnet in relation to the ferromagnetictube in such a manner, that a part of the magnet is inside the tube.Alternatively the magnet can be kept still and the ferromagnetic tube ismoved in relation to the magnet. The magnet is attached to a bar, thatcan be ferromagnetic or is not ferromagnetic, and by means of which themagnet can be moved in the ferromagnetic tube.

The magnet can have the shape of a round bar or a peg, but it can alsohave another shape. The magnetising axis of the magnet may also vary.The magnetising axis can be either longitudinal, whereupon it isparallel to the longitudinal axis of the bar and the poles of the magnetare at the ends of the bar. Then the magnetising is parallel to theferromagnetic tube, i.e. parallel to the direction of movement of themagnet or the tube.

However, the magnetising axis of the magnet can also be transverse,whereupon it is perpendicular in relation to the longitudinal axis ofboth the ferromagnetic tube and the bar-like magnet. Then the directionof magnetisation is transverse to the direction of movement of themagnet or the tube.

On the other hand the magnet can also consist of several separatemagnets, that can be alike or different and that can be attached toeach. other by means of magnetic force or through a material, that isferromagnetic or non-ferromagnetic. The magnet may also be a combinationof magnetic and ferromagnetic material. The magnet may also be either apermanent magnet or an electrical magnet.

By means of the magnet arrangement, protective membrane and the vesselsto be used, microparticles can be treated very efficiently in both largeand small liquid volumes. Focusing the micoparticles to the very tip ofthe magnet unit enables both concentrating from large volumes andtreating microparticles in small volumes. Indeed, a universal solutionfor applications including microparticles both on large and small scaleis described in the invention.

The invention presents that by designing the form of the outer surfaceof the plastic shield or the elastomer in a particular manner sufficientsupport is achieved to collect the mass of microparticles to becollected around the shield in a preferable and reliable manner. Theterm particular design of form refers to, for example, grooves, cavitiesand/or protuberances of different sizes and depths. When gatheringbetween these formations, the pellet of microparticles gets particularsupport from the shield, while the magnet unit is moved against liquidcurrents. The effect produced by viscose samples is very significant,which means at worst, that microparticles do not stay attached to oneside of the shield, but stay in solution. The above-described form hasnaturally a great benefit to the collecting reliability, when handlinglarge volumes.

The invention describes a magnet unit, by means of which microparticlesmay be collected in many different applications. The essential technicalsolution in the invention is the possibility to, by means of aferromagnetic tube, regulate the force and the adjustment of themagnetic field to the surrounding protective membrane, around of whichthe microparticles are collected. The magnet can be moved in and out inrelation to the ferromagnetic tube, whereupon the magnetic field of themagnet is changed. While the magnet is out, a magnetic field equal tothe amount of magnet outside of the ferromagnetic tube is adjusted tothe protective shield. Then microparticles can be collected outside ofthe protective shield. When the magnet is completely moved inside theferromagnetic tube, there is no considerable magnetic field adjustingoutwards. In this case the microparticles do not gather around theprotective membrane, but stay in solution. The tube can be solid oradjustable in order to achieve the best possible efficiency forcollecting. The magnet and the ferromagnetic tube/sleeve according tothe invention are in use also in the extra-vessel solution, whereuponcollecting of microparticles to the inner surface of the vessel iscontrolled by means of the magnet.

The microparticles may contain affinity ligands, enzymes, antibodies,bacteria, cells or cell organelles. Binding of the desired componentscan also be brought about by choosing the surface properties of themicroparticles to be used and the composition of the buffers in anappropriate, preferable manner in order to bind the desired componentsfrom the samples. Examples include ion exchange, hydrophobic and reversephase chromatography. Then, for example, binding and releasing ofproteins from the surface of the microparticles is performed by means ofappropriately chosen buffers and solutions. For example, salt contentand pH value are then very important factors.

An affinity ligand may be, for example, a one- or two-strandednucleotide sequence, such as, for example, DNA (Deoxyribonucleic Acid),RNA, mRNA or cDNA (Complementary DNA) or PNA (Peptide Nucleic Acid), aprotein, a peptide, a polysaccharide, an oligosaccharide, a smallmolecular compound or a lectin. An affinity ligand may also be one ofthe following: Ovomucoid, Protein A, Aminophenyl Boronic Acid, ProcionRed, Phosphoryl Ethanolamine, Protein G, Phenyl Alanine, Proteamine,Pepstatin, Dextran sulfate, EDTA (Ethylenediaminetetraacetic Acid), PEG(Polyethylene Glycol), N-acetyl-glucosamine, Gelatin, Glutathione,Heparin, Iminodiacetic Acid, NTA (Nitrilotriacetic Acid), Lentil Lectin,Lysine, NAD (Nicotinamide Adenine Dinucleotide), Aminobenzamidine,Acriflavine, AMP, Aprotinin, Avidin, Straptavidin, Bovine Serum Albumin(BSA), Biotin, Concanavalin A (ConA) and Cibacron Blue.

Immobilising an enzyme or an affinity ligand means, that an enzyme or anaffinity ligand is attached to the surface of the particles or that itis captured inside a “cage-like” particle, however in such a manner,that the surrounding solution gets in contact with it.

Attaching the enzyme or the affinity ligand to the microparticles can bedone by means of a covalent binding, for example, by means of the aminoand hydroxyl groups in the carrier. Alternatively the binding can bebrought about by means of a bioaffinity pair, for example, abiotin/streptavidin pair. According to one way the enzyme to beimmobilised is produced with DNA technology, for example, in Escherichiacoli bacteria and a particular enzymatic tail has been prepared to theenzyme. This affinity tail binds to microparticles, to which acomponent, binding strongly to the affinity tail in question, isattached in an appropriate manner. The affinity tail may be a smallmolecular compound or a protein. With this arrangement microparticlescould be efficiently utilised while purifying the desired enzyme and, atthe same time, the enzyme bound to the microparticles would be readilyimmobilised on the surface of microparticles to be used in the methoddescribed in the invention.

Attaching of the enzyme or the affinity ligand may also be unspecific,non-covalent, such as adsorption.

The definition “microparticle” refers herein to particles that have arecommended size of 0.10-100 μm. A microparticle can also be aremarkably larger particle, for example, a particle that has a diameterof several millimeters. In the invention microparticles are magnetic,such as, for example, para-, superpara- or ferromagnetic, or consist ofmagnetisable material, or the microparticles are attached to a magneticor magnetisable piece. Microparticles to which, for example, affinityligands or enzymes may be attached, are captured in the vessel by meansof a magnet unit soaked in the vessel, the microparticles are washed inthe same vessel, the opening of the vessel may be closed, the solutionand the microparticles may be mixed by stretching the protectivemembrane consisting of elastomer material, the magnet unit is possiblytransferred to another vessel and the microparticles are released by theaction of the magnet in various appropriate ways as described in theinvention. Alternatively the microparticles do not need to beparticularly liberated from the magnet unit.

The magnet by means of which the particles are captured, can either be apermanent magnet or an electrical magnet. The shape of the magnets mayvary depending on the application. The magnetic field can be differentin the magnets: a longitudinally magnetised magnet, a magnet magnetisedalong the diameter of the magnet or several magnetic poles in the samemagnetic piece. Individual magnets may also be joined to each other bymeans of appropriate ferromagnetic or non-ferromagnetic spacers.

The protective membrane may consist of inelastic material, such as, forexample, polypropylene, polystyrene, polycarbonate, polysulfone andpolyethylene. The protective membrane may also consist ofnon-ferromagnetic metal or ferromagnetic metal. The protective membranemay also consist of stretchy elastomer material, such as, for example,silicone rubber, fluoroelastomer, polychloroprene, polyurethane orchlorosulfonated polyethylene. The protective membrane may also betreated with particular agents and thereby altering the properties ofthe protective membrane. The protective membrane may thus be coatedwith, for example, teflon (PTFE, polytetrafluoroethylene). It isparticularly important to be able to select the protective material andthe possible additional treatment in such a manner, that the resultenables action according to the invention even with very strong orcorrosive chemicals. The protective membrane may also be designed insuch a manner that it enables the protection of the separate magnetunits, for example in devices containing 8, 12 or 96 channels. The shapeof the protective membrane may be that of a tube, of a plate or it canbe irregularly designed. There are particularly many alternatives whenusing an elastomer protective membrane, because then the magnet insideand the ferromagnetic tube may also give a shape to the protectivemembrane.

A preferred alternative to the protective membrane is an even orplate-shaped protective membrane consisting of stretchy material. Such aprotective membrane may be an individual stretchy membrane in aparticular frame. The frame is intended for to facilitate the use of theprotective membrane and to bring about properties suitable forstretching the membrane. Another alternative is a roll-like embodiment,whereupon the protective membrane may be changed by simply rolling newprotective membrane from a roll. Also this alternative may include theuse of a frame, a specific support or a prop in the case where theprotective membrane is being stretched during the actual use. The use ofsuch a protective membrane, consisting of one plate, is a recommendedalternative when consumption of material is desired to be avoided in theisolation and washing events. The use of a protective membrane that hasthe shape of a plate is also economically more advantageous than the useof protective membranes of large size that have been prepared anddesigned with molding tools.

The use of a plate-like protective membrane in an automated device is avery simple and efficient alternative. When using a plate-likeprotective membrane, an initial stretching may be performed in the firststage by means of a ferromagnetic sleeve. At this stage the magnet isstill inside the ferromagnetic sleeve and there is no magnetic fielddirected to the microparticles outside the protective membrane. At thesame time as the protective membrane is further maintained stretched,the magnet may be brought out of the ferromagnetic sleeve in anappropriate manner. Then the magnet stretches the protective membraneadditionally and brings about a gathering of microparticles around theprotective membrane at a spot, where the magnetic pole/poles are. Bymoving the magnet in or out of the sleeve, the solution in the tube ismixed by means of the magnet. Mixing can also be performed by moving theferromagnetic sleeve up and down.

The embodiment presented above is particularly preferable when treatingmicroparticles in small vessels, such as, for example, in microplates,that have, for example, 96, 384 or 1536 wells. The presented way ofmixing the solution and the microparticles is preferred, because thewhole device does not need to be moved. Mixing is brought about bysolely moving the magnet and/or the ferromagnetic sleeve. The presentedapproach is particularly preferable for the reason, that no conventionalshakers are needed in the process and the vessels may be simultaneouslyclosed. Conventional shakers are not able to efficiently mix smallamounts of solution and particularly not to keep the microparticles insolution. A great problem for the known devices and methods is the rapidsedimentation of the microparticles on the bottom of the well.

In the above mentioned known microplates, where small liquid volumes areused, the evaporation of the liquid during incubations and mixings is acritical issue. By using the protective membrane in the presented manneraccording to the invention, the microparticles may also be treated insmall volumes, because the protective membrane closes the opening of thewell at the same time, whereby the evaporation of the liquid decreases.Therefore no separate lid consisting of aluminum, rubber or gummed tapeis according to the invention needed to cover the microplates duringmixings and incubations.

Particularly when separate protective membranes are used in the transferdevices, the protective membrane may be designed in a specific manner inits tip. The design of the tip may be intended to bring about thetransfer of an amount as large as possible of microparticles in areliable manner, for example, from a viscous biological sample toanother vessel. While gathering large amounts of microparticles to thetip of the oblong protective membrane, as is the case when using alongitudinally magnetised permanent magnet, the outer layers ofmicroparticles continuously risk to be liberated and stay in thesolution. Also the turgor at the interface of the solution and the airis very strong and brings about a similar effect causing themicroparticles to be liberated.

The protective membrane may indeed be designed in such a manner, thatthe microparticles stay attached as well as possible to the protectivemembrane while moving the transfer device despite of the emergingcurrents and despite of the penetration of the liquid surface and theeffect of the surface tension on the liquid surface. Therefore variousniches and protuberances can be made to the protective membrane, wherebya reliable transfer of the collected microparticles to another solutionis brought about. Then the protective membrane may consist either ofstretchy or inelastic material.

The protective membrane made of stretchy material may have a particulardesign, that assures the reliable collection and transfer of a largeamount of microparticles from a vessel to another. For this purpose theedges of the protective membrane may have particular protuberances andniches, where the microparticles gather. Then it is preferable to use atransversely magnetised magnet by means of which microparticles can becollected over a broad area. By designing the protective membraneparticular structures supporting microparticle masses are brought about.The design also influences the disturbing effects of liquid currents andliquid tension. When using stretchy material and spots of varyingthickness, the protuberances and niches of the protective membrane arestretched in various ways. This phenomenon can be efficiently utilisedboth in releasing microparticles and particularly in bringing about anefficient mixing in the solution.

In the presented manner the protective membrane itself acts as anelement that brings about mixing and is thereby a very efficient devicefor performing the mixing. Most preferably the design of the protectivemembrane varies in different spots of the protective membrane. Whenmicroparticles are desired to be collected from the solution, the magnetis moved downwards and the membrane is simultaneously stretched. Whilestretching the protective membrane, the specific design of its surfacebrings about the gathering of microparticles to the sheltered andsupporting areas on the surface of the protective membrane. When themicroparticles are desired to be removed from the protective membrane,the magnet is moved upwards into the ferromagnetic sleeve. In order tosecure the release of the microparticles the ferromagnetic sleeve maysimultaneously be moved downwards, whereupon the protective membrane isstretched, and then again upwards and repeating these movements in anappropriate manner. In one embodiment a specific mixing unit consists ofa stretchy protective membrane and a movable bar inside the protectivemembrane. There does not need to be a magnet, but its function is tostretch and loosen the protective membrane from within in order to bringabout an efficient mixing in the solution. Such a mixing unit may beused simultaneously to close the opening of the vessel.

A very efficient mixing is brought about in the liquid in the vessel atthe same time, because the appropriate form of the surface of theprotective membrane acts like an underwater wobble pump. Alternativelyit is possible to move the magnet downwards and thereby stretch theprotective membrane, when an efficient mixing is desired to be broughtabout based on the previously described phenomenon. Moving the magnetinstead of the ferromagnetic tube simultaneously brings about alsomoving of the microparticles towards the magnet and the surface of theprotective membrane, that further enhances the mixing. These previouslymentioned methods for mixing a liquid may also be combined in anappropriate manner. This kind of a method for mixing works also whenusing a longitudinally magnetised magnet.

The ferromagnetic tube described in the invention may also be anindividual tube, a set of several tubes together or an arrangement,where individual tubes form a specific formation of tubes. In oneembodiment of the invention the ferromagnetic tube may be a specificferromagnetic plate, that has one or several holes, where one or severalmagnets may move. Such an arrangement is particularly preferred whenusing small volumes, for example, 8-, 24-, 48-, 96- and 384-well plateformats, such as microplates and the like.

According to the invention there may also be an approach, which includesa separate magnet unit for collecting microparticles and a specificdevice or bar for moving the liquid surface in an appropriate mannerdescribed in the invention. This approach enables solutions, where themagnetic bars do not move at all, but the moving of the liquid and themicroparticles is seen to by means of an organ particularly designed forthis purpose. The vessel used in such an approach or the reactor isdesigned in an appropriate manner to meet the needs described herein.

In one embodiment according to the invention there are several separatemagnet units, which all include their own protective membrane. Thesemagnet units may be grouped in an appropriate formation, such as, forexample, fan-like in line, along the arc of a circle or several arcs ofa circle within each other, whereby each bar gather an appropriateamount of particles around it.

The device and the method according to the invention are not limited to,for example, molecular biology or purification of proteins, but they aregenerally applicable in fields, where ligands bound to microparticlescan be used to synthesize, bind, isolate, purify or enrich desiredbiological components from various samples: diagnostic applications,biomedicine, enrichment of pathogens, synthesis of chemicals, isolationof poisons, viruses, bacteria, yeasts and cells. Also analyticalmethods, immunoassays and DNA hybridisation methods are within the scopeof the invention.

Method Application of the Invention

The device and the method according to the invention are applicable tobe used in very many application areas, for example, protein chemistry,molecular biology, microbiology, cell biology and proteomics. Theinvention has applications in the industry, diagnostics, analytics andresearch.

For purifying proteins there is a need for purification experiments insmall volumes and, on the other hand, to increase the capacity to evenvery large volumes. By means of the described invention proteinpurifications may be done, when the need arises, from various samplevolumes. Protein chemists need to be able to purify protein from asample that has been pre-treated as little as possible, such as, forexample, cell lysates. It is also important to change the capacity ofpurification according to ever changing needs. At present it is possibleby changing the column sizes to be used. As the purification proceeds,enrichment of the protein is-one of the essential operations. Inpractice this means decreasing the liquid volume without any significantloss or denaturation of proteins. At present the most widely usedmethods include dialysis or filtration. Both of these methods require alot of time. By means of the device and the method described in thisinvention a versatile method, that is applicable for varying samplevolumes, can be provided to the field of proteins. Changing the capacityis easy without buying or preparing new columns. For a larger samplevolume a larger amount of microparticles is simply chosen and after theprotein has been bound, microparticles and protein are collected out ofthe solution by means of the device and the method described in theinvention. The washing steps can be performed either in the same vesselor by changing the vessel. In the previous case the washing buffers usedneed to be conducted out of the vessel and replaced with a fresh washingbuffer. Changing the buffer may also be done by means of various valveor aspiration arrangements. After the washes the proteins bound to themicroparticles may be released to a small volume and the proteinsolution may be efficiently enriched.

When the need arises, decreasing the volume can be performed in stagestowards a smaller volume.

By means of the device and the method described in the invention, forexample, ion exchange chromatography, reverse phase chromatography,hydrophobic chromatography and affinity chromatographic purificationscan be made. Also gel filtration can be accomplished with the describeddevice, but it requires performing the gel filtration in a column andthereafter collecting the microparticles by means of a device accordingto the invention and outflow of the proteins to a small volume. Themethod enables, for example, removing salt from samples without largelyincreasing the volume compared to classical gel filtration columns.

The use of immobilised enzymes to process various proteins, sugars, fatsand various so called biopolymers is a very important application areafor the described invention. An important characteristic compared to theuse of soluble enzymes is the possibility to easily reuse theimmobilised enzymes. Washing the immobilised enzyme by means of thedescribed invention for further use is very easy and efficient.

Examples for essential groups of enzymes and individual enzymes, usedfor example in the industry, include:

-   -   CARBOHYDRASES: Alpha-Amylases, Beta-Amylase, Cellulase,        Dextranase, Alpha-Glucosidase, Alpha-Galactosidase,        Glucoamylase, Hemicellulase, Pentosanase, Xylanase, Invertase,        Lactase, Pectinase, Pullulanase    -   PROTEASES: Acid Protease, Alkaline Protease, Bromelain, Ficin,        Neutral Proteases, Papain, Pepsin, Peptidases, Rennin, Chymosin,        Subtilisin, Thermolysin, Trypsin    -   LIPASES AND ESTERASES: Triglyceridases, Phospholipases,        Esterases, Acetylcholinesterase, Phosphatases, Phytase,        Amidases, Aminoacylase, Glutaminase, Lysozyme, Penicillin        Acylase    -   ISOMERASES: Glucose Isomerase, epimerases, racemases    -   OXIDOREDUCTASES: Amino Acid Oxidase, Catalase, Chloroperoxidase,        Glucose Oxidase, Hydroxysteroid Dehydrogenase, Alcohol        dehydrogenase, Aldehyde dehydrogenase, Peroxidases    -   LYASES: Acetolactate Decarboxylase, Aspartic Beta-Decarboxylase,        Fumarase, Histidase, DOPA decarboxylase    -   TRANSFERASES: Cyclodextrin Glycosyltransferase,        Methyltransferase, Transaminase, Kinases    -   LIGASES    -   PHOSPHATASES: Alkaline Phosphatase

The use of enzymes is very common in many branches of the industry, someexamples of which follow: the synthesis and modification of lipids,proteins, peptides, steroids, sugars, amino acids, medicines, plastics,fragrances, chemicals and so called chiral chemicals.

Various synthesizing and cleaving enzymes associated to glycobiology,such as, for example, endo- and exo-glycosidases, are also within thescope of the invention. Enzymes familiar from applications of molecularbiology, such as restriction enzymes, nucleases, ribozymes, polymerases,ligases, reverse transcriptases, kinases and phosphatases are alsowithin the scope of the method described in the invention. As examplesof DNA/RNA modifying enzymes the following can be mentioned: CIAP (CalfIntestinal Alkaline Phosphatase), E. Coli alkaline phosphatase,exonucleases (for example, P1 nuclease, S1 nuclease), ribonucleases,RNases (e.g. Pancreatic RNase, RNase H, RNase T1, RNase M, RNase T2),DNA ligases, RNA ligases, DNA polymerases, Klenow enzyme, RNApolymerases, DNA kinases, RNA kinases, terminal transferases, AMVreverse transcriptase and phosphodiesterases. The use of these and otherDNA/RNA modifying enzymes is very polymorphous both in the research andapplications of molecular biology. Proteases are very important enzymesin proteomics and protein chemistry, example of which include trypsin,chymotrypsin, papain, pepsin, collagenase, dipeptidyl peptidase IV andvarious endoproteinases. Synthetic enzymes, catalytic antibodies andmulti-enzyme complexes may be used in the ways described in theinvention. The use of the invention is neither limited by the use ofenzymes and other catalytic components in water-free conditions, forexample in organic solvents.

As concrete examples of embodiments of the invention in the field ofmolecular biology the following may be mentioned:

Cloning of the DNA Inserts:

For cloning the DNA inserts restriction enzymes are needed, (e.g. EcoRI, Hind III, Bam HI, Pst I, Sal I, Bgl II, Kpn I, Xba I, Sac I, Xho I,Hae III, Pvu II, Not I, Sst I, Bgl I), creating blunt ends (e.g. heatstable polymerases, Klenow Fragment DNA Polymerase I, Mung Beannuclease), ligations (e.g. T4 DNA Ligase, E. coli DNA Ligase, T4 RNALigase), phosphorylation (e.g. T4 Polynucleotide Kinase),dephosphorylation (e.g. CIAP, E. coli Alkaline Phosphatase, T4Polynucleotide Kinase) and deletions (e.g. T4 DNA Polymerase, heatstable polymerases, Exo III Nuclease, Mung Bean Nuclease).

Synthesis and Cloning of the cDNA:

Reverse Transcriptase, RNase H, DNA polymerase I, T4 DNA polymerase I,E. coli DNA Ligase.

Labeling of Nucleic Acids:

5′ labelling (e.g. T4 Polynucleotide Kinase), 3′ addition (e.g. T4 RNALigase), 3′ fill-in (e.g. Klenow Fragment DNA Polymerase I, T4 DNAPolymerase), 3′ exchange (e.g. T4 DNA Polymerase, heat stablepolymerases), nick translation (e.g. E. coli DNA Polymerase I, heatstable polymerases), replacement synthesis (e.g. T4 DNA Polymerase, heatstable polymerases, Exo III Nuclease), random priming (e.g. KlenowFragment DNA Polymerase I, heat stable polymerases) and RNA probes (e.g.T7 RNA Polymerase, SP6 RNA Polymerase).

Sequencing of Nucleic Acids:

Sequencing of DNA (e.g. E. coli DNA Polymerase I, Klenow Fragment DNAPolymerase I, heat stable polymerases) and Sequencing of RNA (e.g.Reverse Transcriptase, heat stable Reverse Transcriptases).

Mutagenisation of Nucleic Acids:

Oligonucleotide directed (e.g. T4 DNA Polymerase, T7 DNA Polymerase,heat stable polymerases) and Misincorporation (e.g. Exo III Nuclease,Klenow Fragment DNA Polymerase I, heat stable polymerases).

Mapping:

Restriction (e.g. Exo III Nuclease), Footprinting (e.g. Exo IIINuclease) and Transcript (e.g. Reverse Transcriptase, Mung BeanNuclease).

Purification of Nucleic Acids:

Isolation and purification of genomic DNA, PCR fragments, DNA/RNA probesand plasmid DNA.

DNA Diagnostic Techniques:

DNA Mapping, sequencing of DNA, SNP analyses (Single NucleotidePolymorphism), chromosome analyses, DNA libraries, PCR (Polymerase ChainReaction), Inverse PCR, LCR (Ligase Chain Reaction), NASBA (Nucleic AcidStrand-Based Amplification), Q beta replicase, Ribonuclease ProtectionAssay.

DNA Diagnostics:

RFLP (Restriction Fragment Length Polymorphism), AFLP (AmplifiedFragment Polymorphism), diagnostics of bacterial infections, bacterialresistance for antibiotics, DNA fingerprints, SAGE (Serial Analysis ofGene Expression) and sequencing of DNA.

The method described for culturing and isolating cells may be broadlyutilised. Cells of interest include, for example, stem cells, Blymphocytes, T lymphocytes, endothelial cells, granulocytes, Langerhanscells, leucocytes, monocytes, macrophages, myeloid cells, natural killercells, reticulocytes, trophoblasts, cancer cells, transfected cells andhybridoma cells. Commonly known methods, such as, for example, a director indirect isolation method, may be used in isolation of cells. In thefirst one, the direct isolation method, the desired cells are separatedby binding them to the surface of microparticles by utilising, forexample, specific antibodies. In the indirect method, not the desiredcells, but all the other cells are bound to the microparticles. Thedesired cells stay in this case in the solution.

The method described in the invention applies well to the culture,isolation, purification and/or enrichment of bacteria, viruses, yeastsand many other uni- or multi-cellular organisms. A particularlyimportant application area is the enrichment of pathogenic bacteria,such as, for example, Salmonella, Listeria, Campylobacter, E. Coli O157and Clostridium, viruses, parasites, protozoans or other small organismsfrom a large liquid volume. The device and the method described in theinvention can be exploited also within these application areas.

Biocatalysis commonly refers to the use of bacteria, enzymes or othercomponents containing enzymes in the process. Enzymes or bacteria can beimmobilised to a suitable solid carrier and the agent being treated isbrought into connection with the immobilised components by using, forexample, classical columns. According to this invention cells or enzymescan be attached to microparticles in an appropriate manner, whichmicroparticles may then be used according to the invention to performvarious enzymatic reactions.

Also analytical methods, immunoassays and DNA hybridisation methods asexamples for research and routine tests are within the scope of theinvention.

Isolation of cell organelles and various cell fractions is also withinthe scope of the application area of the invention. Cell organelles maybe purified in a normal manner by utilising, for example, specificantibodies and various affinity ligands.

There are different needs to purify nucleic acids, starting from thepurification of tiny amounts of DNA (Deoxyribonucleic Acid), RNA(Ribonucleic Acid) or mRNA (Messenger RNA) from large volumes of severalliters. The method according to the invention can be used to isolatenucleic acids from both large and small sample volumes efficiently.

By means of the method a chain can be formed between culturing/growing,isolation and purification events according to various needs. Thedesired cells may, for example, first be isolated from the sample andpurified. Thereafter, for example, the cell organelles can be isolatedfrom the cells. The cell organelles are purified and the process maycontinue, for example, with purification of DNA or proteins.Microparticles equipped with various coatings and characteristics can beused in stages during the process. The last stage may be for example,enrichment of the purified product to the desired volume, amplificationand detection of the product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a partially sectioned view of an embodiment of themagnet unit equipped with another kind of protective membrane.

FIG. 2 corresponds to FIG. 1 and presents the action of the magnet unitin another stage.

FIG. 3 corresponds to FIG. 1 and presents the action of the magnet unitin the third stage.

FIG. 4 corresponds to FIG. 1 and presents the action of the magnet unitin the fourth stage.

FIG. 5 presents a partially sectioned view of yet another embodiment ofthe magnet unit equipped with another kind of a protective membrane.

FIG. 6 presents schematically a sectioned view of several parallelmagnet units, that have a mutual plate-like protective membrane.

FIG. 7 corresponds to FIG. 6 and presents parallel magnet unitsaccording to a second embodiment.

FIG. 8 corresponds to FIG. 6 and presents parallel magnet unitsaccording to a third embodiment.

FIG. 9 corresponds to FIG. 6 and presents parallel magnet unitsaccording to a fourth embodiment.

FIG. 10-30 present the action of vessels containing filters, the mixingunit and the magnet unit while treating microparticles.

FIG. 31-40 present the action of a layer of microparticles, vesselscontaining filters and the magnet unit while treating microparticles.

FIG. 41-46 present the closing of vessels containing filters and theaction of the magnet unit while treating microparticles.

FIG. 47-59 present the use of an external magnet and a ferromagneticsleeve as such, together with the mixing unit and the magnet unit whiletreating microparticles.

FIG. 60-71 present the use of an external magnet and ferromagneticsleeve together with the vessel, the mixing unit and the magnet unitwhile treating microparticles.

FIG. 72-82 present the use of an external magnet and ferromagneticsleeve together with the vessel, the mixing unit and the magnet unitwhile treating microparticles.

FIG. 83-94 present various mixing units.

FIGS. 95 and 96 present the use of an external magnet, ferromagneticsleeve and a spring under the magnet while treating microparticles.

FIG. 97-104 present the use of a transversely magnetised magnet and alayer of microparticles in the case, where microparticles are notreleased during the process from the top of the protective membrane.

FIG. 105-112 present the use of a magnet, magnetised along itslongitudinal axis, and a layer of microparticles in the case wheremicroparticles are not released during the process from the top of theprotective membrane.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a magnet unit 10, that includes a transverselymagnetised magnet 13, a ferromagnetic sleeve 12 and a protectivemembrane 21, that contains ridges 29 on its outer surface. Between theridges 29 there are niches, where microparticles 22 gather and by meansof which reliable collection of a large amount of microparticles tobroad surfaces and their transfer from one vessel to another is assured.

FIG. 2 presents the magnet unit 10 of FIG. 17 in a position, where themagnet 13 is pushed completely out of the ferromagnetic sleeve 12. Thenthe transversely magnetised magnet 13 collects microparticles 22 to itsprotective membrane 21 with its whole length. When pushing the magnet 13out, the protective membrane 21 thereby stretches in such as manner,that large niches or pockets will form between ridges 29. Themicroparticles 22 will stay in these pockets in such a manner, that itis easy to keep them still in place while lifting the magnet unit 10.The liquid currents caused by the movement of the magnet unit 10 and thedisturbing effect of surface tension caused by penetration of thesurface do not release microparticles 22 from the pockets.

FIG. 3 presents a situation, where the magnet 13 is pushed completelyout of the ferromagnetic sleeve 12 and simultaneously the ferromagneticsleeve 12 is also pushed completely out. Then the ferromagnetic sleeve12 pushed on top of the magnet 13 neutralises the magnetic force of themagnet 13 and the microparticles 22 are released from the protectivemembrane and transferred to the liquid.

FIG. 4 again presents a situation, where only the ferromagnetic sleeve12 is completely pushed out. In this case the magnet 13 does neitherhave magnetic force, and so the microparticles 22 do not gather on thesurface of the protective membrane 21. This stage presented in FIG. 26can instead be used by turns with the stage in FIG. 23, whereby anefficiently mixing pump effect is brought about in the liquid. Also thestages in FIGS. 18 and 19 can naturally be used by turns, that is, whilethe magnet 13 is completely pushed out, only the ferromagnetic sleeve 12is moved back and forth. A mixing pump effect is achieved also in thismanner in the liquid.

FIG. 5 presents another magnet unit 10, that includes a longitudinallymagnetised magnet 13, a ferromagnetic sleeve 12 and a protectivemembrane 21, that has a pocket 42 at its end for microparticles 22. Bymeans of such a structure a large amount of particles 22 may becollected, which particles do not easily get released from the surfaceof the protective membrane 21 during the transfer.

FIG. 6 presents a vertical section of a microplate 82, which has severalwells 83. There are several parallel magnet units 10 on top of themicroplate 82, that have a common plate-like protective membrane 21. Theprotective membrane 21 consists of stretchy material, whereby the samemembrane may be used jointly for the adjacent magnet units 10. Themembrane is most preferably taken from a roll, whereupon it also iseasily changeable.

FIG. 7 presents two parallel magnet units 10 a and 10 b, that have acommon protective membrane 21. In the exemplary device of FIG. 7 theaction of the magnet units 10 a and 10 b is at different stages. Theferrometallic sleeves 12 a and 12 b of both the magnet units 10 a and 10b are pressed against the protective membrane 21 in such a manner, thatthe protective membrane 21 is pressed against the edges 84 of the wells83 of the microplate 82 thereby closing and sealing the wells 83 withthe membrane 21. The magnet of the magnet unit 10 b is additionallypushed downwards-towards the well 83 of the microplate 82 in such amanner, that the protective membrane 21 and the head of the magnet 13 binside of it are in the liquid 23. Then the microparticles 22 in theliquid 23 gather in the end of the transversely magnetised magnet 13 bon top of the protective membrane 21.

FIG. 8 presents an embodiment, where the magnet units 10 a and 10 b donot contain separate ferrometallic sleeves. They are replaced by aferrometallic plate 12, which is designed in such a manner, that thereare juts projecting downwards right by the wells of the microplate.Magnets 13 a and 13 b are placed in the openings right by the juts ofthe ferrometallic plate 12. In FIG. 8 the action of the magnets 13 a and13 b of the magnet units 10 a and 10 b are at different stages in thesame way as in FIG. 7.

FIG. 9 presents an embodiment, where the magnet units 10 a and 10 b alsohave a common ferrometallic plate 12 replacing the sleeves, which platein this case is a straight plate. Magnets 13 a and 13 b are placed inthe openings of the ferrometallic plate 12. Also in this figure themagnets 13 a and 13 b of the magnet units 10 a and 10 b are at differentstages. As distinct from the solution in FIG. 7, the protective membrane21 is pressed against the edges 84 of the wells 83 of the microplate 82by means of magnets 13 a and 13 b and not by means of ferrometallicsleeves. The magnet 13 a of the magnet unit 10 a is in the sealingposition, while the magnet 13 b of the other magnet unit 10 b is in theposition for collecting microparticles.

FIG. 10-26 present stepwise a solution according to one embodiment,whereby the microparticles 22 in the vessel 26 are fetched by means ofthe magnet unit 10, that contains a protective membrane 21 consisting ofelastomer material, such as, for example, silicone rubber. FIG. 10presents a vessel 26, that contains appropriate microparticles 22 in asolution 23, which solution 23 and microparticles 22 may have beenincubated appropriately in order to bind the desired biologicalcomponents from the sample in the solution 23.

FIG. 11 presents a magnet unit 10, inside of which there is atransversely magnetised magnet 13 and the magnet 13 may be moved in theferromagnetic sleeve 12. Such a magnet unit is brought into a vessel 26and the microparticles 22 gather on the surface of the protectivemembrane 21 at the very spot, where the magnet 13 is outside theferromagnetic sleeve 12. The protective membrane 21 may also have ridges29, between of which the microparticles 22 may very well settle down.The protective membrane 21 is stretched by means of the magnet 13,whereupon the distance between the ridges 29 in the protective membraneis also increased and more area for collecting is available.

FIG. 12 presents a situation, where the microparticles 22 collected tothe magnet unit 10 may be transferred away from the solution 23 byremoving the magnet unit 10 away from the vessel 26.

In FIG. 13 the magnet unit 10 is brought into a vessel 76 including afilter, which vessel contains appropriate liquid 23, such as, forexample, appropriate wash buffers. The filter 77 may consist of variousmaterials and it may have various thicknesses and the porosity grade maygreatly vary according to different needs. In place of the filter 77there may be a membrane or a specific solution including valves. In thevessel 76 including a filter, the magnet 13 of the magnet unit 10 isbrought upwards inside the ferromagnetic sleeve 12, whereupon there isno magnetic field left around the protective membrane 21 and themicroparticles 22 may be released from the surface of the protectivemembrane 21 into the solution 23.

In FIG. 14 the ferromagnetic sleeve 12 is used to stretch and loosen theprotective membrane 21 appropriately in turns and thus currents arebrought about inside the solution 23. It is possible by means of themethod described in the invention to both efficiently mix the solutionand further the release of the microparticles 22 from the surface of theprotective membrane 21.

In FIG. 15 the microparticles 22 are collected from the vessel 76including a filter by bringing the magnet 13 of the magnet unit 10 outof the ferromagnetic sleeve 12 and by stretching the elastomerprotective membrane 21 in an appropriate manner. In FIG. 16 the magnetunit 10 is lifted from the vessel 76 including a filter. Thebackward-and-forward stretching of the protective membrane 21 broughtabout by means of the ferromagnetic sleeve 12 and the collecting ofmicroparticles 22 by means of the magnet 13 may also be appropriatelydone in turns, in case very efficient mixing properties are desired tobe brought about. In FIG. 17 the removing of the solution from thevessel 76 through the filter 77 on its bottom via the channel 85 joinedto the bottom of the vessel 76 by means of an aspiration/vacuum unit,which unit is not presented in the figure, is presented.

In FIG. 18 appropriate new solution 23 is added to the vessel 76containing the filter 77. If there is an appropriate space in the vessel76, the magnet unit 10 does not need to be removed from the vessel 76while adding the next solution 23. The magnet unit 10 can thereby beappropriately placed, for example, by one wall of the vessel 76including a filter during the addition of the solution 23. In the nextstep a new solution 23 is added to the vessel including a filter.

In FIG. 19 the magnet unit 10 is brought back to the vessel 76 includinga filter, to which vessel a new solution 23 is added. These bufferchanges and collecting of microparticles may be performed with thevessel including a filter consecutively as many times as is desired. Bymeans of an approach obtained in this manner the amount of disposablesto be used may be greatly decreased, because multiple washes andincubations may be performed in the same vessel. FIG. 19 presents therelease of microparticles 22 from the magnet unit 10 to the solution 23,as is described in FIG. 13.

In FIG. 20 the microparticles 22 are mixed by means of the magnet unit10 in the solution 23 as previously described in FIG. 14. In FIG. 21 themagnet 13 of the magnet unit 10 is transferred outside the ferromagneticsleeve 12 and the microparticles 22 are collected from the solution 23on top of the protective membrane 21, as is described in FIG. 14. InFIG. 22 the magnet unit 10 and the microparticles 22 collected on top ofthe protective membrane 21 are transferred away from the vessel 76including a filter. In FIG. 23 the magnet unit 10 is transferred to anew vessel 26. In FIG. 24 the microparticles 22 are released to thesolution 23 in the manner presented in FIG. 14.

FIG. 25 presents a situation, where the magnet unit 10 is removed fromthe vessel 26 and the microparticles 22 released from the top of theprotective membrane 21 are in the solution 23. The process may becontinued from this vessel when the need arises. Finally themicroparticles 22 may be totally removed from the vessel and thecomponents bound on the surface of the microparticles 22 in thebeginning of the process from the vessel 26 presented in FIG. 10 may bereleased to the solution.

FIG. 26 presents another way of proceeding from the situation describedin FIG. 19. In FIG. 26 microparticles 22 are mixed in the solution 23 bymeans of the magnet unit 10 in the vessel 76 including a filter as ispreviously described in FIG. 14. In FIG. 27 the magnet unit 10 istransferred away from the vessel 76 including a filter and themicroparticles 22 stay in the solution 23. FIG. 28 presents theaspiration of the solution 23 away from the vessel 76 containing thefilter 77, for example, through the channel 85 joined to the bottom ofthe vessel by means of an aspiration or vacuum device. Themicroparticles 22 do not go along the solution, but stay on top of thefilter 77. FIG. 29 presents the addition of the following solution tothe vessel 76 including a filter.

FIG. 30 further presents the removal of the solution added in theprevious figure in the manner described in FIG. 28. Finally thecomponents bound to the microparticles 22 may be released and collectedto a separate vessel placed in the vessel 76 containing the filter. Allthe removals done by means of the vessel 76 including a filter may beperformed either by using a vacuum or a centrifuge. Additions of thesolution 23 done to the vessel 76 including a filter may be performed bymeans of ordinary devices for liquid handling, such as manual pipettesor dispensers. Also automated devices for liquid handling may be appliedin the method.

FIG. 31-40 present stepwise an approach according to one embodiment ofthe invention, whereby the microparticles 22 are in the vessel 76containing the filter 77, in which vessel the removal of solutions andthe addition of the following solution may be repeated many times. InFIG. 31 the microparticles 22 are in the solution 23 in the vessel 76including a filter. In FIG. 32 the solution 23 is aspirated from thevessel 76 including a filter and the microparticles 22 stay on top ofthe filter 77 to form a layer of microparticles 78.

The following solution that may be, for example, a sample, is brought tothe vessel 76 including a filter in FIG. 33. The solution may be furtheraspirated from the vessel 76, whereupon the desired components are boundon the surface of the microparticles 22 in the layer of microparticles78. These steps may be repeated many times and finally an appropriateamount of solution 23 is added, which solution is not aspirated from thevessel 76 including a filter.

In FIG. 34 a magnetic tool 10 is brought into the vessel 76 including afilter. By moving the ferromagnetic sleeve 12 and the magnet 13 in anappropriate manner, the stretching and loosening of the protectivemembrane 21, and thereby an efficient mixing in the solution 23, isbrought about. There may be appropriate ridges 29 on the surface of theprotective membrane 21 in order to increase the efficiency of mixing andto further collecting of microparticles 22. By mixing the solution 23,the homogenisation of the layer of microparticles 78 on top of thefilter 77 to the solution 23 is brought about. In FIG. 34 the protectivemembrane 21 is presented in its stretched form and in FIG. 35 theprotective membrane 21 is presented in its non-stretched or shrunkenform.

In FIG. 36 the transversely magnetised magnet 13 of the magnet unit 10is brought out of the ferromagnetic sleeve 12 and the magnet 13stretches the protective membrane 21 in an appropriate manner. Themicroparticles 22 from the solution 23 gather on top of the protectivemembrane 21. In FIG. 37 the magnet unit 10 is removed from the vessel 76including a filter together with the collected microparticles 22 on topof the protective membrane 21. In FIG. 38 the magnetic tool 10 and themicroparticles 22 are transferred to the new vessel 28, that containsthe new solution 23.

In FIG. 39 the microparticles 22 are released from the surface of theprotective membrane 21 by moving the ferromagnetic sleeve 12 on top ofthe magnet 13. The release of the microparticles 22 may be enhanced byappropriately stretching the protective membrane 21 by means of theferromagnetic sleeve 12 or by moving the entire magnetic tool 10 in anappropriate manner.

In FIG. 40 the magnetic tool 10 is transferred away from the vessel 26and the microparticles 22 are released to the solution 23. It ispossible to proceed from this step forward and release, for example, thecomponents bound from the original sample to the microparticles 21 tothe solution 23. If the microparticles cause disturbance in the extendedapplications, the microparticles may be removed from the solution, ifnecessary. There may be various numbers of additions of the solutionand/or the sample described previously to the vessel 76 including afilter. The number of treatments performed in the vessel 26 may alsovary when the need arises. There may be various amounts ofmicroparticles 22, whereupon their binding capacity may be greatlyincreased or decreased when the need arises. A particularly importantapplication is to run a large volume of sample through the layer ofmicroparticles 78, whereupon the desired sample is attached on thesurface of the microparticles 22. There may be specific ligands, suchas, for example, antibodies, peptides or nucleotides bound on thesurface of the microparticles 22. There may be a biological component,such as, for example, bacteria, viruses, cells, nucleic acids, proteinor peptide in the sample, which component is desired to be collected onthe surface of the microparticles 22. The components bound on thesurface of the microparticles 22 may finally be released to a smallvolume. This is particularly applicable to the case where there is verylittle of the component to be collected, such as bacteria, in a greatvolume of sample.

After treatment and possible washes, the microparticles are collectedfrom the filter by means of the magnet unit. There may preferably be atransversely magnetised magnet in the magnet unit and an appropriatelydesigned protective membrane, that together enable the collecting ofeven great masses of microparticles from the filter. By stretching theelastomer protective material according to the invention, a mixing ofthe solution is brought about, whereupon the microparticles gathered onthe filter may be well liberated from the filter. By means of the magnetunit the microparticles may be further transferred to other vessels forpossible further washing, incubating, eluating, amplifying and/ordetecting.

The invention is not limited to the use of an elastomer protectivemembrane and a transversely magnetised magnet, but to the use of amagnetic tool in general together with a vessel including a filteraccording to the method. There does not need to be any separateprotective membrane on top of the magnet.

FIG. 41-46 present stepwise an approach according to one embodiment, inwhich approach the use of more than one magnet unit and vessels 76including a filter for treating microparticles, such as mixing,collecting, releasing and transferring, closing and opening vessels aswell as handling solutions, such as removing and adding solutions, ispresented. The magnet units and the vessels containing filters may be,for example, arranged in a row of 8 or 12 units. A very convenientapplication method may also be 24-, 48-, 96- and 384-well plates,whereupon the treatment of samples is remarkably faster.

Such an approach to treat microparticles is particularly applicable toan automated device, that includes the necessary dispenser for handlingliquids, a vacuum work station (e.g. 96 and a 384-well format) forvessels containing filters and a multimagnet magnetic unit (for example,containing 8, 12 or 96 magnets).

FIG. 41 presents the closing of the vessel 76 including a filter bymeans of magnet units 10 a and 10 b by moving the magnet unit 10 and themicroparticles 22 to the vessel 76 including a filter. In FIG. 42 themicroparticles 22 are released from the top of the protective membrane21 to the solution 23 by moving the magnet 13 into the ferromagneticsleeve 12. In FIG. 43 the protective membrane is stretched by moving theferromagnetic sleeve 12 and thus an efficient mixing is brought about inthe solution. An important property is that the magnet unit 10 does notmove entirely, but only the stretching movement of the protectivemembrane 21 in the solution is brought about by means of theferromagnetic sleeve 12. The protective membrane 21 seals the vesselsimultaneously with the mixing of the solution. In FIG. 44 the magnet 13of the magnet unit 10 is moved outside the ferromagnetic sleeve 12 andit stretches the protective membrane 21. The microparticles 22 gatherfrom the solution 23 on top of the protective membrane 21. In FIG. 45the magnet unit 10 and the microparticles 22 are removed from the vessel76 including a filter and the solution is also aspirated from thevessel. In FIG. 46 the following solution is added to the vessel 76including a filter and the process may continue according to FIG. 41-45.

The protective membrane of the magnet unit 10 may have a specific designto increase the tightness of the junction between the protectivemembrane 21 in the magnet unit 10 and the vessel 76 including a filter.The simplest way to tighten the junction is to press the magnet unit 10tightly against the vessel 76 including a filter. The stretchingproperties of the elastomer material in the protective membrane 21 maybe utilised to close the vessel tightly and to open it easily. Solutions23 may be changed several times when the need arises by simplyaspirating the previous solution through a filter before adding a newone to the vessel.

FIG. 47-59 present stepwise an approach according to one embodiment, inwhich approach FIG. 47 presents a vessel 26 containing microparticles22. The microparticles 22 may be coated with an appropriate ligand andthere may be an appropriate sample in the solution 23, of which sample agiven biological component is desired to be bound on the surface of themicroparticles 22. In FIG. 48 the vessel 26 is moved beside the magnet13 outside the vessel 26, whereupon the microparticles 22 gather to forma layer of microparticles 78 in the vicinity of the magnet 13 on theinner surface of the vessel. FIG. 49 presents a situation, where thesolution 23 is aspirated from the vessel 26 in such a manner, that themicroparticles 20 stay on the surface of the vessel 26. In FIG. 50 thefollowing solution 23 is added to the vessel 26 and the magnetic fieldis removed by moving the ferromagnetic sleeve 12 on top of the magnet13. When the magnetic field is absent, the microparticles 22 may bebrought to a homogenised state in the solution 23 by mixing the solution23 in different ways.

In FIG. 51 the microparticles 22 are homogenised from the layer ofmicroparticles 78 to the solution 23, for example, by means of the tipof a pipette by moving the solution 23 back and forth in the vicinity ofthe layer of microparticles 78. In FIG. 52 the ferromagnetic sleeve 12is removed from around the magnet 13, whereupon the magnetic field drawsthe magnetic particles to form a pellet. Thereafter the solution 23 isaspirated and the following solution is added. These intermediate stepsmay be repeated when the need arises depending on the application.Compared to the conventional method, this method does not require movingthe vessel or the magnet physically far away from each other.

In FIG. 53 the magnetic tool 10 described in the invention is brought tothe solution 23 in the vessel 26 and the protective membrane 21 isstretched downwards by means of the ferromagnetic sleeve 12. In FIG. 54the ferromagnetic sleeve 12 is moved upwards and the stretching of theprotective membrane 21 is decreased in the solution 23. By repeatedlystretching and loosening the elastomer protective membrane 21 in such amanner, that the magnet 13 is not outside the ferromagnetic sleeve 12, agood mixing is obtained and the microparticles 22 are homogenised fromthe layer of microparticles 78 to the solution 23. Stretching theprotective membrane 21 is performed by means of the ferromagnetic sleeve12. At the same time as the solution 23 is being mixed by stretching theprotective membrane 21, the vessel 26 may be tightly closed by means ofthe magnet unit 10 and the protective membrane 21 and thus theevaporation of the solutions from the vessel 26 may be inhibited. Thisembodiment is particularly preferred in the case, where long incubationsand/or high temperatures are desired to be used, whereupon theevaporation becomes a considerable problem.

In FIG. 55 the microparticles 22 are collected by means of the magnetunit 10 on the top of the protective membrane 21 by moving the magnet 13out of the ferromagnetic sleeve 12. In FIG. 56 the microparticles 22 aretransferred together with the magnet unit 10 from the vessel 26. Thesolution 23 may be aspirated from the vessel 26 and the followingsolution and the microparticles 22 may be brought back to the samevessel 26. Another way is to transfer the microparticles 22 to anothervessel, where the microparticles 22 may also be released when the needarises.

In FIG. 57 a specific mixing tool is brought to the vessel 26 and theprotective membrane 21 is in its stretched form. The basic principle ofthe mixing tool 80 in relation to the mixing effect is the same as ispresented in FIGS. 53 and 54, i.e. by stretching the elastomerprotective membrane 21 back and forth, currents are brought about in thesolution and simultaneously the opening of the vessel is closed. In thiscase there is no magnet in the mixing tool 80, but a specific bar 11inside the protective membrane 21, by moving of which bar the protectivemembrane consisting of elastomer material may be stretched and loosenedwhen the need arises.

FIG. 58 presents the mixing tool when the protective membrane is in itsloosened form and a homogenised solution 23 of the microparticles 22 isobtained. In FIG. 59 the ferromagnetic sleeve 12 is removed from aroundthe magnet 13 outside the vessel 26, whereupon the microparticles form alayer of microparticles 78 on the inner surface of the vessel. Themixing tool 80 is removed from the vessel 26 or it is in the vessel 26even when the solution 23 is aspirated from the vessel 26 and thefollowing solution is added to the vessel 26.

FIG. 60-71 present stepwise an approach according to one embodiment, inwhich approach treating a set of vessels containing filters, such as,e.g. a 96-format plate, and microparticles 22 together with the magnetunit 10 and the mixing tool 80. In FIG. 60 there is a magnet 13 underthe set of vessels containing filters and between the separate vessels76, around of which magnet there is the ferromagnetic sleeve 12 that maybe moved in relation to the magnet 13. The microparticles 22 are in thesolution 23 in the vessels 76 containing filters.

In FIG. 60 there is the ferromagnetic sleeve 12 on top of the magnet 13and the magnetic field is switched off. In FIG. 61 the ferromagneticsleeve 12 is moved from around the magnet 13, whereupon the magneticfield of the magnet 13 gathers the microparticles 22 to form a layer ofmicroparticles 78 on the inner surface of the vessels 76. In FIG. 62 thesolution 23 may be aspirated through the filter bottom 77, for example,by means of a vacuum device and the layer of microparticles 78 staysattached to the inner surface of the vessel 76 including a filter bymeans of the magnetic field of the magnet 13.

In FIG. 63 the following solution 23 may be added to the vessel 76including a filter and the ferromagnetic sleeve 12 is again moved on topof the magnet 13. In FIG. 64 there is no longer a magnetic fieldprojected to the layer of microparticles 78, but the layer ofmicroparticles 78 can not be homogenised to the solution 23 withoutmixing. In an embodiment of the invention both the magnet 13 and theferromagnetic sleeve 12 may be separately moved. Then the spot, wherethe microparticles 22 gather, and the area of this spot may becontrolled simply by means of the magnet 13 and the ferromagnetic sleeve12. FIG. 60-63 present an embodiment, where the magnet 13 does not move,but only the ferromagnetic sleeve 12 moves in relation to the magnet 13.

In FIG. 65 a specific mixing tool is brought into the vessel 76including a filter, which tool has an elastomer protective membrane 21and a bar 11, that may be moved up and down inside the protectivemembrane 21. By moving the bar 11 downwards, the protective membrane 21is stretched and in FIG. 65 the protective membrane 21 is presented inits stretched form. In FIG. 66 the bar 11 is moved upwards and thetension of the protective membrane 21 is restored. By means of such amixing, liquid currents are brought about in the solution 23 and themethod is particularly well suited for mixing microparticles 22 in smallvolumes, such as, e.g. in 96-well plates. The mixing tool 80 itself doesnot move during this event, but only the bar 11 inside the protectivemembrane 21 is moving. In this case the mixing tool 80 and theprotective membrane 21 in it may close the vessel 76 including a filterduring the mixing. While the mixing tool 80 is mixing, the magnetoutside the vessel 76 including a filter is completely covered with theferromagnetic sleeve 12.

In FIG. 67 the ferromagnetic sleeve 12 is moved away from around themagnet 13 outside the vessel 76 including a filter and themicroparticles 22 may be collected to form a layer of microparticles 78on the inner surface of the vessel 76 including a filter. The mixingtool 80 is removed from the vessel 76 including a filter. After thisstep the solution 23 may be aspirated through the filter 77 and thefollowing solution may be added to the vessel 76 including a filter. Ifthe opening of the vessel 76 including a filter does not need to beclosed during the mixing step, the protective membrane of the mixingtool 80 does not need to consist of elastomer material and it ispossible that it does not need to be a protective membrane at all. Inthis case mixing is performed by means of a rod, a bar or a pegconsisting of only plastic or other suitable material.

In FIG. 68 the magnet unit 10 is brought into the vessel 76 including afilter, on top of which unit there is a protective membrane 21, which isstretched to bring about mixing in the manner presented in FIGS. 65 and66, in which case the magnet 13 is constantly inside the ferromagneticsleeve 12. In FIG. 68 the protective membrane 21 is in its stretchedform. The ferromagnetic sleeve 12 acts as the factor stretching andloosening the protective membrane 21. Also in this case the magnet unit10 and the protective membrane 21 may close the opening of the vessel 76including a filter during mixing and other events. In FIG. 69 theprotective membrane is in its loosened form and the microparticles 22are homogenised in the solution 23.

In FIG. 70 the microparticles 22 are collected on the top of theprotective membrane 21 by moving the magnet 13 out of the ferromagneticsleeve 12 and by stretching the protective membrane 21 by means of themagnet 13. In FIG. 71 the magnet unit 10 and the microparticles 22collected on top of the protective membrane 21 are removed from thevessel 76 including a filter. The solution 23 may be aspirated throughthe filter 77 and the following solution may be added to the vessel 76including a filter. The microparticles 22 may be brought by means of themagnet unit 10 back into the vessel 76 including a filter and mixingsand collectings described previously may be performed when the needarises. The microparticles 22 may also be brought to another vessel forfurther treating.

FIG. 72-82 present stepwise an approach according to an embodiment, inwhich approach treating a set of vessels containing filters, such as,e.g. a 8-, 24-, 48- or 96-well plate, and microparticles 22 togetherwith the magnet unit 10 and the mixing tool 80 is presented. In FIG. 72there is a magnet 13 under the set of vessels containing filters andbetween the separate vessels 76, around of which magnet there is theferromagnetic sleeve 12 that may be moved in relation to the magnet 13.The microparticles 22 are in the solution 23 in the vessels 26. In FIG.72 there is a ferromagnetic sleeve 12 on top of the magnet 13 and themagnetic field is switched off.

In FIG. 73 the ferromagnetic sleeve 12 is moved away from around themagnet 13, whereupon the magnetic field of the magnet 13 collects themicroparticles 22 to the inner surface of the vessels 26 to form a layerof microparticles 78. In FIG. 74 the solution 23 may be aspirated, forexample, by means of washers or pipettes and the layer of microparticles78 stays attached to the inner surface of the vessels 26 by means of themagnetic field of the magnet 13. In FIG. 75 the following solution 23may be added to the vessel 26 and the ferromagnetic sleeve 12 is movedagain on top of the magnet 13. In an embodiment of the invention boththe magnet 13 and the ferromagnetic sleeve 12 may be separately moved.Then the spot, where the microparticles 22 gather and the area of thisspot may be controlled simply by means of the magnet 13 and theferromagnetic sleeve 12. FIG. 72-75 present an embodiment, where themagnet 13 does not move, but only the ferromagnetic sleeve 12 moves inrelation to the magnet 13.

In FIG. 76 a specific mixing tool is brought into the vessel 26, whichtool has an elastomer protective membrane 21 and a bar 11, that may bemoved up and down inside the protective membrane 21. By moving the bar11 downwards the elastomer protective membrane 21 is stretched and inFIG. 76 the protective membrane 21 is presented in its stretched form.In FIG. 77 the bar 11 is moved upwards and the tension of the protectivemembrane 21 is restored. By means of such a mixing liquid currents arebrought about in the solution 23 and the method is particularly wellsuited for mixing microparticles 22 in small volumes, such as, e.g. in96- and 384-well plates. The mixing tool 80 itself does not move duringthis event, but only the bar 11 inside the protective membrane 21 ismoving. In this case the mixing tool 80 and the protective membrane 21in it may close the vessel 76 including a filter during the mixing.While the mixing tool 80 is mixing, the magnet outside the vessel 26 iscompletely covered with the ferromagnetic sleeve 12.

In FIG. 78 the ferromagnetic sleeve 12 is moved away from around themagnet 13 outside the vessel 26 and the microparticles 22 may becollected to form a layer of microparticles 78 on the inner surface ofthe vessel 26. The mixing tool 80 is removed from the vessel 26. Afterthis step the solution 23 may be aspirated and the following solutionmay be added to the vessel 26. If the opening of the vessel 26 does notneed to be closed during the mixing step, the protective membrane of themixing tool 80 does not need to consist of elastomer material and it ispossible that it does not need to have a protective membrane at all. Inthis case mixing is performed by means of a rod, a bar or a pegconsisting of only plastic or other suitable material. The rods, barsand pegs used for mixing may be disposable and they may be, for example,in 96-format, if a 96-well plate is being mixed.

In FIG. 79 a magnet unit 10 is brought into the vessel 26, on top ofwhich unit there is a protective membrane 21, which is stretched tobring about mixing in the manner presented in FIGS. 76 and 77, in whichcase the magnet 13 of the magnet unit 10 is constantly inside theferromagnetic sleeve 12 of the magnetic tool 10. In FIG. 79 theprotective membrane 21 is in its stretched form. The ferromagneticsleeve 12 of the magnet unit 10 acts as the factor stretching andloosening the protective membrane 21. Also in this case the magnet unit10 and the protective membrane 21 may close the opening of the vessel 26during mixing and other events.

In FIG. 80 the protective membrane 21 is in its loosened form and themicroparticles 22 are homogenised in the solution 23. In FIG. 81 themicroparticles 22 are collected on the top of the protective membrane 21by moving the magnet 13 of the magnet unit 10 out of the ferromagneticsleeve 12 and by stretching the protective membrane 21 by means of themagnet 13. In FIG. 82 the magnet unit 10 and the microparticles 22collected on top of the protective membrane 21 are removed from thevessel 26. The solution 23 may be aspirated and the following solutionmay be added to the vessel 26. The microparticles 22 may be brought bymeans of the magnet unit 10 back into the vessel 26 and mixings andcollectings described previously may be performed when the need arises.The microparticles 22 may also be brought to another vessel for furthertreating.

FIG. 83-94 present stepwise an approach according to yet anotherembodiment. FIG. 83 presents a mixing tool 80, that has a protectivemembrane 21. The protective membrane 21 has different ridges 29 and theprotective membrane 21 is in its stretched form in the solution 23.There is a bar 11 inside the protective membrane 21, which bar may bemoved upwards and downwards. The bar 11 may consist of differentmaterials (for example, plastic or metal). Currents may be brought aboutin the solution 23 when stretching the protective membrane 21 bypressing the bar 11 downwards and loosening the stretch by moving thebar 11 upwards. Currents may be further enhanced when using differentridges 29 on the surface of the protective membrane 21 and by choosingthe vessel 26 appropriately.

In FIG. 84 protective membrane 21 is in its loosened form. In FIGS. 83and 84 the opening of the vessel 26 is closed by means of the mixingtool 80 in order to decrease evaporation and minimise the risk forsplashing. Closing the vessel 26 may be performed during the mixing,because only the bar 11 inside the mixing tool 80 is moving, therebystretching the protective membrane 21 consisting of elastomer material.Such an approach is particularly efficient in automated devices and whenmixing small volumes.

FIG. 85 presents an approach, where the protective membrane 21 is solidfor adjacent mixing tools 80 and vessels 26 containing solution 23. Theprotective membrane 21 is stretched and loosened by means of the bar 11.In FIG. 85 the protective membrane 21 is in its loosened form and theprotective membrane 21 solid for vessels 26 closes the opening of thevessels 26. In FIG. 86 the protective membrane 21 is in its stretchedform and the bar 11 is pressed downwards. A particularly preferredembodiment is, for example, one solid protective membrane, for example,a plate consisting of silicone rubber on top of the microtiter plate,such as, for example, 96-, 384-, 1536-well plates. Also in this approachthe closing of the wells may be arranged simultaneously while thesolutions in the wells are being mixed. By means of such-an embodimenteither all wells may be mixed or only desired wells may be mixed whilethe other wells may be left unmixed. A particularly preferred embodimentare reactions and incubations performed in high temperature, wherebyevaporation is considerable. By closing the vessels tightly evaporationmay be remarkably reduced.

FIG. 87-94 present different mixing tools as pairs, where the other oneis in its stretched and the other one in its loosened form. The figurespresent different alternatives for the design of the protective membrane21. An appropriate design of the protective membrane is also dependenton the inner measures and ridges of the vessel as well as the amount ofsolution to be used in the vessel.

FIGS. 95 and 96 present stepwise an approach according to an embodiment,where a possible approach is presented for the use of an external magnetand a ferromagnetic sleeve, for example, with 96-well plates. There isthe transversely magnetised magnet 13 and the ferromagnetic sleeve 12 inFIG. 95. In addition there may be a spring suspension 81 under themagnet. By pressing the vessel 26, for example, a 96-well platedownwards, the vessel 26 simultaneously presses the magnet 13 inside theferromagnetic sleeve 12 and the string 81 under the magnet 13 tightens.When the magnet 13 is inside the ferromagnetic sleeve 12, there is nomagnetic field outside and the microparticles 22 stay in the homogenisedsolution 23.

In FIG. 96 the vessel 26 is no longer pressed down, but due to thestring 81 the magnet 13 comes out of the ferromagnetic sleeve 12 and themagnetic force of the magnet 13 affects the adjacent vessels 26. Themicroparticles 22 in the vessels gather in the vicinity of the magnet 13to form a layer of microparticles 76 on the inner surface of the vessel.

Pressing the vessel 26 may be performed, for example, by means of themagnet unit 10, described in the invention, or the mixing tool 80. Themixings described in the invention and the closing of the vessel 26 maybe performed efficiently by means of such a string approach. By pressingthe vessel 26 downwards, for example, by means of the magnet unit 10,the openings of the vessel are closed and simultaneously the externalmagnet 13 is pressed inside the ferromagnetic sleeve 12. When the vessel26 is kept down by means of the magnet unit 10, the mixings described inthe invention and the homogenisations, collections and transfers may beperformed efficiently. Simultaneously the vessel 26 stays surely closed.The vessels 26 may also have filters on the bottom and the advantagesdescribed in the invention for using filter-bottom vessels crop outclearly also in this approach. The described string 81 does not need tobe a string at all, but the movement of the magnet may be arranged bymeans of a specific motor, whereby the vessel is not needed at all forpressing the magnet 13 inside the ferromagnetic sleeve 12. The motorisedversion enables moving both the magnet and the vessel in use.

FIG. 97-112 present stepwise an approach according to yet anotherembodiment, in which the use of microparticles 22 on top of theprotective membrane 21 of the magnet unit 10 throughout the wholeprocess, such as, for example, when performing nucleic acid or proteinpurifications, immunoassays and DNA hybridisation assays. Themicroparticles 22 may be coated with appropriate ligands (for example,with antibodies, oligonucleotides and peptides) to bind desiredbiological components from the solution/sample. It is essential, thatthe microparticles 22 are collected in the beginning of the process onthe surface of the magnet unit, as is described in FIGS. 97 and 105, andthat the microparticles are not released in the various wash steps orincubation steps, depending on the application, of the process. By meansof the described method a particular, active reaction surface may beestablished by collecting the microparticles 22 by means of the magnet13 on the surface of the protective membrane 21. The microparticles arenot lost during the wash steps and incubation steps, because themicroparticles 22 are not released during the process. Finally ameasurement may be performed either in the solution 23 or in the layerof microparticles 78.

FIG. 97 presents a vessel 76 including a filter, in which vessel thereis solution 23 containing microparticles 22. In FIG. 98 the magnet unit10 is brought into the solution 23 in the vessel 76 including a filterand the microparticles 22 are collected on the surface of the protectivemembrane 21 to form a particular layer of microparticles 78. Theprotective membrane 21 may consist of elastomer or non-elastomermaterial. The magnet 13 presented in the figure is transverselymagnetised, the ferromagnetic sleeve 13 is not on top of the magnet 13.By using the transversely magnetised magnet 13 the microparticles 22 maybe collected over a very large area around the protective membrane 21.Such an approach is particularly preferred, when a very large area forcollecting and reaction kinetics are needed, for example, in sensitiveimmunoassays. The layer of microparticles 78 may be washed in the vessel76 including a filter by aspirating and adding appropriate solutions tothe vessel including a filter. The additions and aspirations ofsolutions in the vessel 76 including a filter may be performed, forexample, by means of aspiration/vacuum devices and dispensers.

In FIG. 99 the magnet unit 10 is removed from the vessel 76 including afilter to another vessel 26 containing a new solution 23. The layer ofmicroparticles 78 is not homogenized to the solution 23, but the magnetunit 10 may be appropriately moved in the solution 23. In the vessel 26also particular incubations may be performed by letting the magnet unit10 and the protective membrane 21 be in the solution 23. The magnet unit10 and the protective membrane 21 may appropriately close the opening ofthe vessel 26 and prevent evaporation of the solution 23. In FIG. 100the magnet unit 10 is removed from the vessel 26 and the layer ofmicroparticles 78 is on top of the protective membrane 21.

FIG. 101 presents the removal of the solution 23 from the vessel 26, forexample, by means of a washer or a pipette. In FIG. 102 the addition ofa new solution to the same vessel 26 is described. In FIG. 103 themagnet unit 10 is transferred to the vessel 23 now containing a newsolution 23. The layer of microparticles 78 is not homogenised to thesolution 23, but the layer of microparticles 78 may be moved and letstand in the solution 23 for an appropriate period of time. In FIG. 104the magnet unit 10 and the layer of microparticles 78 on top of theprotective membrane 21 are removed from the vessel 26. The solutions maybe changed in the described process as many times as needed and finallya measurement for determining the concentration of the isolatedbiological component may be performed in the solution 23 in the vessel26. Alternatively, the concentration of the biological component may bedetermined in the layer of microparticles 78.

FIG. 105 presents a vessel 76 including a filter, in which vessel thereis solution 23 containing microparticles 22. In FIG. 106 the magnet unit10 is brought into the solution 23 in the vessel 76 including a filterand the microparticles 22 are collected on the surface of the protectivemembrane 21 to form a particular layer of microparticles 78. Theprotective membrane 21 may consist of elastomer or non-elastomermaterial. The magnet 13 presented in the figure is magnetised along thelongitudinal axis of the magnet unit 10, the ferromagnetic sleeve 13 isnot on top of the magnet 13. By using the magnet 13 magnetised along thelongitudinal axis of the magnet unit 10 the microparticles 22 may becollected precisely around the tip of the protective membrane 21. Suchan approach is particularly preferred, when handling very small volumesof solutions. The layer of microparticles 78 may be washed in the vessel76 including a filter by aspirating and adding appropriate solutions tothe vessel 76 including a filter. The additions and aspirations ofsolutions in the vessel 76 including a filter may be performed, forexample, by means of aspiration/vacuum devices and dispensers.

In FIG. 107 the magnet unit 10 is removed from the vessel 76 including afilter to another vessel 26 containing a new solution 23. The layer ofmicroparticles 78 is not homogenised to the solution 23, but the magnetunit 10 may be appropriately moved in the solution 23. In the vessel 26also particular incubations may be performed by letting the magnet unit10 and the protective membrane 21 be in the solution 23. The magnet unit10 and the protective membrane 21 may appropriately close the opening ofthe vessel 26 and prevent evaporation of the solution 23. In FIG. 108the magnet unit 10 is removed from the vessel 26 and the layer ofmicroparticles 78 is on top of the protective membrane 21. FIG. 109presents the removal of the solution 23 from the vessel 26, for example,by means of a washer or a pipette.

In FIG. 110 the addition of a new solution to the same vessel 26 isdescribed. In FIG. 111 the magnet unit 10 is transferred to the vessel23 now containing a new solution 23. The layer of microparticles 78 isnot homogenised to the solution 23, but the layer of microparticles 78may be moved and let stand in the solution 23 for an appropriate periodof time. In FIG. 112 the magnet unit 10 and the layer of microparticles78 on top of the protective membrane 21 are removed from the vessel 26.The solutions may be changed in the described process as many times asneeded and finally a measurement for determining the concentration ofthe isolated biological component may be performed in the solution 23 inthe vessel 26. Alternatively, the concentration of the biologicalcomponent may be determined in the layer of microparticles 78.

According to the invention, one embodiment for the method presented inFIG. 97-112 is to use vessels containing all the necessary solutions,microparticles, antibodies, labels, wash buffers and substrates readilydispensed. Vessels containing the previously mentioned solutions mayalso be closed by means of aluminum foil, different stickers orelastomer solutions.

FIG. 97-112 present a way of producing and using the layer ofmicroparticles as an expansive solid-phase, that is applicable also withdifferent vessels, such as, for example, tubes and wells. Then byutilising an external magnet and a ferromagnetic sleeve themicroparticles are bound to the inner surface of the vessel to form anappropriate layer. The magnet approach may also be of another kind, suchas, an electric magnet or a regular permanent magnet. The layer ofmicroparticles ought to be relatively thin, whereby the advantagesdescribed in the Invention are achieved. It is also possible to usemixing methods described in this invention with such a vessel and theclosing of the opening of the vessel in the case, where the methods aredesired to be efficiently exploited.

Transferring and using the magnet unit, even when it does not contain anelastomer protective membrane, together with vessels containing filtersand an external magnet, are within the scope of the invention of thepatent. The above mentioned embodiments of the invention serve only asexamples of applying the idea according to the invention. It is evidentfor those skilled in the art that various embodiments may exist withinthe scope of the claims that follow farther behind.

LIST OF REFERENCE NUMERALS

-   10 magnet unit-   11 bar-   12 ferromagnetic tube or sleeve-   13 magnet-   21 protective membrane-   22 microparticles-   23 solution-   25 liquid surface-   26 vessel-   28 rotational axis-   29 ridge of the protective membrane-   40 multi-channel transfer device for microparticles-   41 group of magnet units-   42 pocket-   76 vessel including a filter on its bottom-   77 filter-   78 layer of microparticles-   79 tip of a pipette or a dispenser/washer-   80 mixing device-   81 spring-   82 microplate-   83 well-   84 edge of the well-   85 outlet channel

The invention claimed is:
 1. A device for handling magneticmicroparticles, comprising: (a) a vessel (26), wherein the vessel is atube or a well; (b) a magnetic tool (10), comprising a magnet (13), aprotective shield (21) or coating, a ferromagnetic sleeve (12), whereinthe magnet (13) and the ferromagnetic sleeve (12) are each movable inrelation to each other and wherein the magnetic field of the magnet (13)is increased when the magnet (13) is partly or completely outside theferromagnetic sleeve (12), and a plurality of magnetic microparticles(22) contained within the vessel (26); wherein the magneticmicroparticles (22) are selected from the group consisting of:ferromagnetic, paramagnetic, and superparamagnetic particles.
 2. Thedevice according to claim 1, wherein the protective shield is anelastomer or non-elastomer shield (21) for binding the microparticles(22).
 3. The device according to claim 1, wherein the protective shieldis an elastomeric membrane having bellows covering the magnetic tool. 4.The device according to claim 1, wherein the opening of the vessel (26)is closed by the magnetic tool (10).
 5. The device according to claim 4,wherein the protective shield is an elastomeric membrane.
 6. The deviceaccording to claim 1, comprising a filter (77) at the bottom of thevessel (26).
 7. The device according to claim 6, wherein the bottom ofthe vessel (26) further comprises a channel (85) for conducting solution(23) through the filter (77) out from the vessel.
 8. The deviceaccording to claim 1, wherein the magnet (13) has a magnetizing axisthat is parallel to the longitudinal axis of the ferromagnetic sleeve(12) and the magnet (13).
 9. The device according to claim 1, whereinthe magnet (13) has a magnetizing axis that is transverse to thelongitudinal axis of the ferromagnetic sleeve (12) and the magnet (13).